Understanding the consequences of intratumor heterogeneity in sensitivity to antineoplastic drugs both in regard to the immediate effect on therapy and to longer term development of drug resistance, is of clear importance to effective therapy of human cancer. In order to study these consequences, we have developed a tumor model system, consisting of a series of mouse mammary tumor subpopulation lines and sublines, that are differentially sensitive to chemotherapeutic drugs. One of the major conclusions of our work thus far is that tumor subpopulations may alter each other's sensitivity to therapeutic drugs so that the drug response of a heterogeneous tumor is not simply the "sum" of that of its component parts. We propose to investigate the mechanisms and consequences of this finding by reconstructing heterogeneous tumors using mixing paired subpopulations that differ in chemosensitivity and in ability to interact with each other. Our first aim is to examine the significance of heterogeneity on in vivo sensitivity to single chemotherapeutic drugs. Mechanisms by which heterogeneity can affect drug sensitivity will be elucidated by various in vivo techniques, including manipulation of growth parameters by the use of different tumor cell dosages and/or sites of injection, use of immunosuppressed mice, and use of related drugs which have different known properties (such as trimetrexate vs methotrexate). Interactions will be modeled in vitro so that mechanisms can be further ascertained, using the collagen gel culture system, which mimics the in vivo situation in that cells have maximum ability to interact with each other and display other similarities such as microenvironmental effects and growth/cell cycle characteristics. Our second aim is to analyse the significance of heterogeneity at different treatment times, including treating during primary tumor growth and after surgery as an adjuvant procedure. All tumors which are produced in experiments described above, will be analysed for the presence, physical distribution (zonality) and chemosensitivity of their component tumor subpopulations, using colony forming assays in selective media to identify and quantitate the populations (Aim 3). In order to better understand the basis for decreased drug sensitivity of tumor cells in our in vitro model of tumor cell growth, the collagen culture system, we will focus on differences in recovery from drug treatment in collagen cultures vs monolayer. The importance of factors such as growth rate and cell shape in affecting recovery will be determined (Aim 4).